It has long been known that mammals, when confronted with bacterial or viral infections, exhibit efforts at self-healing which are initiated by a complex physiological network referred to as the immune system. The immune system operates in response to a challenge to the mammal by initially recognizing the presence of a foreign organism or pathogen within the animal""s body. In mammals, this is followed by an attack on the foreign organism by the neutrophils, macrophages and other xe2x80x9ckillerxe2x80x9d cells of the immune system. This immune response functions or is xe2x80x9cturned onxe2x80x9d by a variety of immune system regulators which activate the various aspects of the immune system depending upon the type of insult confronting the subject animal.
A substantial component of the immune system is a group of structurally related glycoproteins, collectively known as immunoglobulins, contained within blood and extra cellular fluids. Five immunoglobulin classes have been identified. Immunoglobulin G (IgG), IgM, IgA, IgD and IgP. The basic structural unit of each immunoglobulin class consists of two pairs of polypeptide chains joined by disulfide bonds. The five classes of immunoglobulins have different biological properties and different distributions in the body. The structure responsible for the biological properties of each immunoglobulin class is located on that part of the immunoglobulin molecule which is unique for each classxe2x80x94the Fc fragment. While some antibodies are produced at all times in normal animals, other antibodies are produced only in response to specific antigenic stimulation (e.g., when pathogenically challenged).
IgG is the major antibody class in normal mammalian systems and forms about 70% of the total immunoglobulin. IgG is evenly distributed between intra- and extra vascular pools. It is the first major antibody of the secondary immune response and belongs to the exclusive antitoxin class. IgG is a monomeric protein which can be divided into four sub-chainsxe2x80x94two heavy chains xe2x80x9cHxe2x80x9d and two light chains xe2x80x9cLxe2x80x9d. Taking the four sub-chains together each IgG molecule consists of one H2L2 unit with a molecular weight of approximately 140,000 Daltons. Molecules of the IgG class are actively transported across the placenta and provide passive immunity to newborns at a time when the infant""s immune mechanisms are not developed.
The remaining four immunoglobulin classes are more narrow components of the immune system.
IgM is the first immunoglobulin class produced by the maturing fetus. IgM does not normally cross the placenta from the mother to fetus, but may be produced actively by the fetus prior to birth, especially if the fetus has been exposed to antigens by infection. IgA is found in relatively small amounts in serum and tissue fluids, but is present in high concentrations in external secretion such as saliva, tears, and bronchial secretions. IgE is also present in very low concentrations and appears to be associated with the histamine response. The last immunoglobulin class, IgD, is present in very low concentrations in secretions. IgD stimulates immature lymphocytes to multiply and differentiate thereby causing the production and secretion of other antibodies. Therefore, all immunoglobulin classes are important in immune system responses.
Modulation of the immune system to effect greater response to foreign agents has been an area of interest for some years. The development of specific antibodies through vaccination has long been utilized to provide mammals with long term immune defense mechanisms to specific microorganism forms.
Ansley, U.S. Pat. No. 5,219,578, Jun. 15, 1993 discloses a caprine serum fraction consisting principally of non-adjuvanted IgG. This IgG fraction is useful as an immunostimulant in mammals when challenged by specified diseases.
Recent efforts in immunology have been directed towards the utilization of immune system regulating molecules, rather than one of the five classes of immunoglobulins, to provide increased immune system activity. It is believed that, through the use of immune regulating or immune modulating molecules, a state of general immune system hyperactivity can be induced which may help combat challenges to the immune system (e.g., pathogenic infection). Such infection may arise from a wound site or may arise from an opportunistic blooming when the host organism is simply deprived of sufficient sleep. It is believed that an induced state of general immune hyperactivity would result in a therapeutic response to the challenge. This might be viewed as the opposite of the vaccination type response that produces a specific long-term immunity. If such a non-specific immune response could be initiated at will it could be utilized to either act alone or in conjunction with a conventional treatment directed towards the etiological agents.
Such a mechanism could be based upon activation of phagocytic cells that are capable of responding to a wide range of infectious agents. It may also be that the T-lymphocytes, which are major mediators of the overall immune response, may act to enhance the operation of non-specific cellular immunity even though the T-lymphocytes themselves are a part of the specific immune response.
The search for agents which potentiate the immune response is a driving force in drug research. Cytokines and cationic peptides are two classes of xe2x80x9crelativelyxe2x80x9d low molecular weight compounds which have shown promise in this area of research. At least nine immuno-defense peptide products are commercially available with annual sales of over $4 billion (Latham, P. W., 1999, Therapeutic peptides revisited, Nature Biotechnology 17:755-757).
Bio-active peptides (such as xe2x80x9ccationic peptidesxe2x80x9d) are emerging as promising alternatives for combating antibiotic-resistant bacteria with minimum inhibitory concentrations reported from 1-100 xcexcg/ml Martin, A, T. Ganz, and R. I. Lehrer, 1995. Defensins and other endogenous peptide antibiotics of vertebrates. J. Leukoc. Biol., 58:128-136; Hancock, R. E. W., 1997, Peptide antibiotics, Lancet, 349:418-422). Cationic peptides range from 16-18 amino acid residues for the protegrins (Ganz, T., and R. Lehrer, 1998. Antimicrobial peptides of vertebrates, Curr. Opi. Immunol., 10:41-44.) to 29-35 residues for mammalian defensins (Sawa, T, and K. Turahashi, 1999, Antimicrobial peptides/proteins application to the therapy of sepsis (article in Japanese), Masui, 48:1186-1193.). Due to a compositional prominence of lysine and arginine, they possess a net positive charge of at least 2, and usually 4, 5, or 6 (Hancock, R. L. W., 1997, Peptide antibiotics, Lancet, 349:418-422).
Interleukin-1 (IL-1), tumor necrosis factor-xcex1 (TNF-xcex1) and interferon (IFN) are three cytokines which participate in the immune response IL-1 is involved in the host""s response to antigenic challenge and tissue injury, and has been shown to increase the resistance of mice to pathogenic organisms such as Listeria, Escherichia coli, and Candida albicans (Czuprynski, C. J., and Brown, J. F., 1987, Recombinant murine interleukin-1xcex1 enhancement of nonspecific antibacterial resistance, Infection and Immunity 55:2061-2065; Cross, A. S., Sadoff, J. C., Kelly, N, Bermton, F., and Gemski, P, 1989, Pretreatment with recombinant murine tumor necrosis factor xcex1/cachectin and murine interleukin 1xcex1 protects mice from lethal bacterial infection, The Journal of Experimental Medicine 169:2021-2027; Pecyk, R. A., Fraser-Smith, E. B., and Matthews, T. R., 1989, Efficacy of interleukin-1xcex2 against systemic Candida albicans in normal and immunosuppressed mice, Infection and Immunity 57:3257-3258.). TNF-xcex1 and xcex3-IFN were able to increase the resistance of mice to Salmonella typhimurium (Morrissey, P. J., and Chamer, K., 1994, Treatment of mice with IL-1 before infection with increases resistance to a lethal challenge with Salmonella typhimurium, The Journal of Immunology 153:212-219). Human a IFN""s have potent antiviral and antiproliferative activities, and are currently being utilized as anticancer or antiviral therapeutic agents (Chang, C. J., Chen, T. T., Cox, B. W., Dawes, G. N., Stemmer, W., Punnonen, J., and Patten, P. A., 1999, Evolution of a cytokine using DNA family shuffling, Nature Biotechnology 17:793-797).
Cationic peptides help defend against the constant assault of moderate numbers of bacteria. Each natural peptide has a broad but incomplete spectrum of activity. The host compensates for this by producing an array of different peptides that together have a broader spectrum of activity, and often work in synergy with one another. A single individual may produce dozens of different peptides and more than 500 natural cationic peptides have been discovered (Hancock, R. E. W., 1999. Host defence (cationic) peptides Drugs 57:469-473).
Bio-active peptides have been found to possess antiviral, antibacterial, antifungal, and wound healing properties (Sanglier, J., Haag, H., Huck, T., and Fehr, T, 1993. Novel bioactive components from Actinomycetes: A short review (1988-1992). Res. Microbiol. 144:633-642; Mizuno, T., Wang, G., Zhang, J., Kawagishi, H., Nishitoba, T., and Li, J, 1995, Reishi, Ganoderma Lucidum and Ganoderma Tsugar. Bioactive substances and medicinal effects, Food Rev. Int. 11:151-166; Hancock, R. E. W., 1999. Host defence (cationic) peptides Drugs 57:469-473). A decameric peptide has even been shown to impede the growth and spread of established tumors (Folkman, J., 1999, Angiogenic zip code, Nature Biotechnology 17:749). It is believed that these xe2x80x9cdefensexe2x80x9d peptides are more general in action than antibodies, and as such, have a broader range of activity (Hancock, 1999). These peptides have low toxicity to most mammalian cells and are therefore candidates for development as therapeutic agents (Maloy, W. L., and U. P. Kari, 1995. Structure-activity studies on magainins and other host defense peptides, Biopolymers (Peptide Science) 37:105-122).
Mammalian species used as food animals are subjected to high stress levels during shipment to processing centers and while awaiting processing. Disease is common during such periods of stress.
In addition to food animals, numerous mammalian species such as dogs, cats and other non-food animals are maintained as pets and are subject to various diseases where an immune stimulant would be desirable.
Equine species are susceptible to diseases which are capable of being beneficially treated using immune stimulants.
More exotic mammals, such as those kept in zoos, are subject to stress related and stress non-related diseases due to the artificial environments in which they live. A nonspecific immunomodulator would be desirable for both prophylactic and ameliorative purposes.
The cost associated with the administration of prophylactic ants and the inherent risk of residues of such drugs, remaining in the edible portions of a food animal make it desirable to minimize the administration of such drugs. A simple and elegant means of accomplishing this is to increase the assertiveness of the mammals own disease fighting immune system.
Therefore, it is an object of the present invention to provide a means for modulating the immune response in mammals afflicted with disease.
Another object of the present invention is to provide a means for enhancing the ability of conventional anti-microbial medicaments by providing a concomitant stimulation of the mammal""s immune response.
Yet another object of the present invention is to provide a means of stimulating the immune response in mammals to heighten the mammals ability at self-healing when a challenged by an infectious agent.
Yet another object of the present invention is to provide a means of prophylactically stimulating the immune response in mammals to heighten the animal""s ability to avoid disease prior to being placed in a high stress environment.
Yet another object of the present invention is to provide a means of stimulating the immune response in mammals to heighten the animal""s ability to respond to sub-clinical disease conditions.
It is also an object of the present invention to non-specifically stimulate the immune system of a mammal by removing and fractionating a portion of the mammals blood, storing the low molecular weight fraction until required and re-introducing the fraction into the donor mammal when required either prophylactically or after a disease state has commenced.
The above and further objects and novel features of the invention will more fully appear from the following description and the examples contained therein.